Abstract
The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase. The structure of the unbound form of this thioesterase has been shown to contain the so-called "hot dog" fold with a large helix packed against a five-stranded anti-parallel beta-sheet. To address the manner in which the enzyme accommodates the substrate within the active site, two inhibitors have been synthesized, namely 4-hydroxyphenacyl-CoA and 4-hydroxybenzyl-CoA. Here we describe the structural analyses of the enzyme complexed with these two inhibitors determined and refined to 1.5 and 1.8 A resolution, respectively. These studies indicate that only one protein side chain, Ser(91), participates directly in ligand binding. All of the other interactions between the protein and the inhibitors are mediated through backbone peptidic NH groups, carbonyl oxygens, and/or solvents. The structures of the enzyme-inhibitor complexes suggest that both a hydrogen bond and the positive end of a helix dipole moment serve to polarize the electrons away from the carbonyl carbon of the acyl group, thereby making it more susceptible to nucleophilic attack. Additionally, these studies demonstrate that the carboxylate group of Asp(17) is approximately 3.2 A from the carbonyl carbon of the acyl group. To address the role of Asp(17), the structure of the site-directed mutant protein D17N with bound substrate has also been determined. Taken together, these investigations suggest that the reaction mechanism may proceed through an acyl enzyme intermediate.
Highlights
The soil-dwelling microbe Pseudomonas sp. strain CBS-3 is capable of surviving on 4-chlorobenzoate as its sole source of
The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase
In light of the structural similarity between this thioesterase and -hydroxydecanoyl thiol ester dehydrase complexed with 3-decynoyl-N-acetylcysteamine, a model of 4-hydroxybenzoyl-CoA was positioned into the thioesterase putative active site [3]
Summary
Enzyme Purification and Crystallization—Wild-type Pseudomonas sp. strain CBS-3 4-hydroxybenzoyl-CoA thioesterase was purified as described previously [7]. Crystals of the D17N mutant protein in complex with the substrate 4-hydroxybenzoyl-CoA were first identified from a sparse matrix screen and “optimized” in batch by macro-seeding into droplets containing 8 –10% polyethylene glycol 5000-O-methyl ether, 200 mM KCl, 100 mM MES2 (pH 6.0), and 1 mM substrate with the protein concentration at 5 mg/ml. X-ray Data Collection and Processing—Prior to x-ray data collection, the wild-type thioesterase crystals in complex with 4-hydroxyphenacylCoA or 4-hydroxybenzyl-CoA were transferred to a cryoprotectant solution composed of 20% polyethylene glycol, 250 mM KCl, 20% ethylene glycol, and 100 mM succinate (pH 5.0) This solution included the relevant CoA inhibitor at a concentration of 5 mM. Where [S] is the total ligand concentration, [E] is the total enzyme concentration, Kd is the apparent dissociation constant of the enzymeligand complex, ⌬F is the observed change in fluorescence intensity, ⌬Fmax is the maximum change in fluorescence intensity, and Fo is the initial fluorescence intensity
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